Recent rapid advances in molecular biology have created more demand for high volume testing based on the need to screen ever larger compound libraries, validate ever increasing numbers of genetic markers and test ever more diversified patient populations. This has led to the development of new array formats, particularly for nucleic acid and protein-protein interaction analysis, which permit parallel processing by performing requisite assays in a “multiplexed” format.
Conventionally, such assays are performed by producing arrays of nucleic acids and antibodies by way of “spotting” or “printing” of aliquot solutions on filter paper, blotting paper or other substrates. However, notwithstanding their widespread current use in academic research for gene expression analysis and protein profiling, arrays produced by spotting have shortcomings, particularly in applications placing high demands on accuracy and reliability and calling for the analysis of a large volume of samples at high rates of throughput In another more recently developed technique, spatially encoded probe arrays are produced by way of in-situ photochemical oligonucleotide synthesis. However, this technology is limited in practice to producing short oligonucleotide probes—as a result, alternative technologies are required for the production of cDNA and protein arrays. In addition, in-situ synthersis precludes rapid probe array customization given the time and cost involved in the requisite redesign of the photochemical synthesis process.
In addition to these inherent difficulties relating to assay performance, spatially encoded arrays produced by spotting or in-situ synthesis conventional methods generally require specialized optical scanning instrumentation to extract data of useable quality. Commercial systems available for this purpose rely on confocal laser scanning—a slow process—even at the typically modest spatial resolution of ˜5 μm—which must be repeated for each signal color.
In order to resolve many of the problems associated with diagnostic and analytical uses of “spotted arrays” of oligonucleotides and proteins (as outlined in “Multianalyte Molecular Analysis Using Application-Specific Random Particle Arrays,” U.S. application Ser. No. 10/204,799, filed on Aug. 23, 2002; WO 01/98765), arrays of oligonucleotides or proteins arrays can be formed by displaying these capture moieties on chemically encoded microparticles (“beads”) which are then assembled into planar arrays composed of such encoded functionalized carriers. See U.S. patent application Ser. No. 10/271,602 “Multiplexed Analysis of Polymorphic Loci by Concurrent Interrogation and Enzyme-Mediated Detection,” filed Oct. 15, 2002, and Ser. No. 10/204,799 supra.
Microparticle arrays displaying oligonucleotides or proteins of interest can be assembled by light-controlled electrokinetic assembly (see, e.g., U.S. Pat. Nos. 6,468,811; 6,514,771; 6,251,691) or by a direct disposition assembly method (previously described in “Arrays of Microparticles and Methods of Preparation Thereof,” filed Jul. 9, 2002; Ser. No. 10/192,352).
To perform nucleic acid or protein analysis, such encoded carrier arrays are placed in contact with samples anticipated to contain target polynucleotides or protein ligands of interest. Capture of target or ligand to particular capture agents displayed on carriers of corresponding type as identified by a color code produces an optical signature such as a fluorescence signal, either directly or indirectly by way of subsequent decoration, in accordance with one of several known methods. The identity of capture agents including probes or protein receptors (referred to herein sometimes also collectively as “receptors”) generating a positive assay signal can be determined by decoding carriers within the array. See U.S. patent application “Multianalyte Molecular Analysis Using Application-Specific Random Particle Arrays” Ser. No. 10/204,799.
These microparticle (“bead”) arrays generally contain a number of spectrally distinguishable types of beads within an area small enough to be viewed by a standard optical microscope. The small footprint and high signal contrast permit “instant” direct (“snapshot”) multicolor imaging of the entire array under a microscope, thereby obviating the need for confocal laser scanning.
Analyzing the images recorded from such random encoded bead arrays involves several steps and issues, as described in U.S. patent application “ANALYSIS, SECURE ACCESS TO, AND TRANSMISSION OF ARRAY IMAGES” Ser. No. 10/714,203. To identify individual receptors scoring positive (and negative) in a given assay, the assay image comprising a pattern of signal intensities recorded from individual beads within the array is compared to a decoding image, taken before (or after) the assay. As described in detail in Ser. No. 10/714,203, this process is performed in an automated fashion by constructing a decoding map comprising groups (“clusters”) of beads of the same type (displaying the same type of receptor). A rapid process for constructing the decoding map (also referred to as the “cluster map”) is disclosed herein which invokes an adaptive template.
Determining the intensities of the individal beads in the assay image allows quantitation of results, for example, in gene expression or protein biomarker analysis. To determine bead signal intensities, the image is divided into segments by constructing a grid such that each field in the grid contains at most one bead—the intensities of the grid field intensities are then recorded. Because the grid finding step is performed on an image that is aligned within the viewing field, a first processing step is that of image rotation.
While the grid finding step can be performed on a bright field image (as described in Ser. No. 10/714,203), it is advantageous to eliminate the recording of a bright field image by instead finding the grid directly for decoding and assay images, typically comprising fluorescence images. In addition, the fluorescence image typically has a substantially higher contrast, that is, the ratio of signal to background. However, several problems must be overcome. First, beads displaying receptors which do not produce an assay signal will not be visible in the fluorescence image, and this generally will lead to the corruption of grid edges, thereby affecting the step of image rotation which relies on the accurate determination of the original image orientation by measuring the orientation of edges. Second, signal sources such as fluorescent beads may be randomly displaced from the center of each grid field, a fact which can affect the intensity recorded from that field.